Antispasmodic Activity of Green-Synthesized Phenylaminojuglones: In Vitro and In Silico Studies

Phenylaminojuglones were synthesized under solvent-free conditions via silica gel-mediated aza-Michael addition between juglone and substituted anilines, namely, aniline, 4-methoxyaniline, and 3,4,5-trimethoxyaniline. Among the synthesized derivatives, phenylaminojuglone (AJ-2) exhibited the most potent spasmolytic activity on rat ileal smooth muscle. Molecular docking studies revealed that AJ-2 exhibits potential binding affinities with multiple targets implicated in smooth muscle contraction, including muscarinic receptors, β-adrenergic receptors, nitric oxide synthase, guanylate cyclase, and the Ca_V1.2 calcium channel. Absorption, distribution, metabolism, excretion, and toxicity predictions indicated that AJ-2, AJ-8, and AJ-11 comply with Lipinski's rule of five and do not present major predicted toxicity liabilities, supporting their pharmacological viability. Functional assays demonstrated that AJ-2 significantly inhibited both pharmacomechanical (ACh-induced) and electromechanical (KCl-induced) contractions. Mechanistic studies suggested that its relaxant effects might be mediated by the activation of β-adrenergic receptors, the NO-sGC-cGMP signaling pathway, and the opening of K_V and K_Ca channels. AJ-2 also inhibited extracellular Ca²⁺ influx, as shown by its interaction with verapamil and attenuation of CaCl₂-induced contractions. These findings support AJ-2 as a promising multifunctional spasmolytic agent derived from green chemistry principles and highlight the pharmacological relevance of phenylaminojuglones as novel modulators of the intestinal smooth muscle tone.